Recent advances in pulsed power technology culminated in engineering of unique devices capable of delivering high-voltage, nanosecond-duration electrical pulses (nsEP) to low-impedance loads such as biological tissues and cell samples. Compared to longer pulses (such as those routinely used for electrostimulation and electroporation), nsEP are distinguished by a steep voltage increase (1012-1014 V/cm per second) and extremely high peak E-field (103-106 V/cm), whereas the total energy deposition into exposed tissue remains low and Joule heating does not exceed a few degrees C. Due to extreme E-field values, nsEP can cause unique bioeffects, such as Ca2+ bursts, lasting inactivation of voltage-gated ion channels, cell swelling and blebbing, "nanoelectroporation" of membranes, necrotic and apoptotic cell death. Combined with the ease of affecting only a limited volume of tissue, nsEP are a promising new therapeutic modality for tissue ablation and solid tumors destruction. First animal trials demonstrated the efficiency of nsEP treatment of inoculated tumors. However, physical and physiological mechanisms leading to cell death after nsEP exposure have been poorly understood, which hinders progress in medical applications of nsEP. Remarkably different nsEP sensitivity of different cell types has not been explained, and it is not known which nsEP parameters (e.g., E- field, pulse rate, absorbed dose) determine the cytotoxic effect. Our preliminary experiments established unexpected similarities of nsEP effects with known effects of both sparsely ionizing radiations (SIRs) and chemical agents that cause oxidative stress. For both these modalities, the principal mechanism of cell death is damage by free radicals, and we hypothesize that this is also the case for nsEP exposure. The proposed study consists of four Specific Aims intended to quantify nsEP cytotoxic effects in different cells and under different physiological conditions, to test the free radical damage hypothesis, and explore the mechanisms and pathways responsible for nsEP-induced cell death. Specific Aim 1: Wide-scale quantitative analysis of cell death dependence on the physical parameters of nsEP treatment, including pulse duration, voltage, dose, the number of pulses, and their repetition rate. Specific Aim 2: Explore the role of physiological conditions of the cell culture (cell cycle phase, growth stage, and differentiation) on the sensitivity to nsEP exposure. Specific Aim 3: Analyze possible involvement of free radical damage mechanism in cell death caused by nsEP exposure. Specific Aim 4: Analyze mechanisms of long-term disruption of plasma membrane ionic conductance by nsEP and its possible role as a primary physiological event that leads to nsEP-induced cell death. PUBLIC HEALTH RELEVANCE This study will be focused on physico-chemical and physiological mechanisms that underlie and determine mammalian cells sensitivity to nanosecond-duration, high-voltage electric pulses (nsEP). Anticipated results will help to quantify, predict, and purposefully modify nsEP sensitivity, assist understanding of mechanisms of nsEP bioeffects, and promote the development of nsEP medical applications, such as tissue ablation and destruction of tumors.